Identification and characterization of extensive intra-molecular associations between 3'-UTRs and their ORFs.
During eukaryotic translation, mRNAs may form intra-molecular interactions between distant domains. The 5'-cap and the polyA tail were shown to interact through their associated proteins, and this can induce physical compaction of the mRNA in vitro. However, the stability of this intra-molecular association in translating mRNAs and whether additional contacts exist in vivo are largely unknown. To explore this, we applied a novel approach in which several endogenous polysomal mRNAs from Saccharomyces cerevisiae were cleaved near their stop codon and the resulting 3'-UTR fragments were tested either for co-sedimentation or co-immunoprecipitation (co-IP) with their ORFs. In all cases a significant fraction of the 3'-UTR fragments sedimented similarly to their ORF-containing fragments, yet the extent of co-sedimentation differed between mRNAs. Similar observations were obtained by a co-IP assay. Interestingly, various treatments that are expected to interfere with the cap to polyA interactions had no effect on the co-sedimentation pattern. Moreover, the 3'-UTR appeared to co-sediment with different regions from within the ORF. Taken together, these results indicate extensive physical associations between 3'-UTRs and their ORFs that vary between genes. This implies that polyribosomal mRNAs are in a compact configuration in vivo.
Eldad
Naama
N
Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Yosefzon
Yahav
Y
Arava
Yoav
Y
D020413:3' Untranslated Regions / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D047468:Immunoprecipitation; D016366:Open Reading Frames; D011061:Poly A / Q000378:metabolism; D011132:Polyribosomes / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000378:metabolism; D012333:RNA, Messenger / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics
D020413:3' Untranslated Regions; D012315:RNA Caps; D012333:RNA, Messenger; D011061:Poly A; D011189:Potassium Chloride